Ischemic brain injury results in part from actions of oxygen free radicals. Superoxide dismutase (SOD) and heme-oxygenase-1 (HO-1) are enzymes that protect against free radical injury. The proposed studies will exploit newly developed methods of manipulating the expression of these proteins to (a) identify the ischemic conditions under which these enzymes are most effective in reducing neuronal death, and (b) identify conditions that can negate or render deleterious the actions of SOD or HO-1. The long term goal of these studies is to provide a rationale for tailoring specific stroke therapies to specific clinical conditions. We hypothesize that (a) increased SOD activity is likely to be most beneficial in the presence of nitric oxide, where SOD activity can limit peroxynitrite production, and (b) that the neuroprotection provided by increased SOD activity can be limited or even outweighed in conditions where H2O2 production exceeds catabolism or where H2O2 reactivity is increased. These hypotheses will be tested using cortical cell cultures prepared from mice with genetically altered SOD activity and exposed to metabolic derangements of ischemia: free radical stress, acidosis, substrate deprivation, and excitotoxicity. Similar experiments will test the hypothesis that HO-1 will reduce neuronal death and free radical injury in cultures exposed to ischemic metabolic derangements that promote iron-catalyzed free radical activity. HO-1 will be induced with heme or by stable transduction of HO-1 in the cell cultures. The role of astrocyte SOD and HO-1 in mitigating neuronal injury will be studied with a novel co-culture system that allows removal and replacement of astrocytes in close contact with the neurons. Studies will also be performed with mice in vivo to confirm results of the cell culture studies and to determine how altered SOD or HO-1 expression affects biochemical measures of oxidative injury after administration of free radical generating compounds. This model will in addition be used to adapt microdialysis methods of assessing brain .OH, nitrite and H2O2 for use in the mouse. These biochemical methodologies will also be applied to other studies in this program.